Fatty acids, C16-C18 (even numbered) and C18 (unsaturated) and Fatty acids, C16-C18 (even numbered) and C18 (unsaturated) methyl esters

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD test guideline 422

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

minor recording errors without any impact on study outcome

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

85049-31-6

Cas Number:

85049-31-6

IUPAC Name:

85049-31-6

Details on test material:

Name: Unsaturated Alkyl Carboxylic Acid Methyl Ester (C10-C18 and C12-C22). The test item is also known as FAME
Batch/Lot No: ASG_161418
Storage Conditions: Ambient room temperature, protect from light/Nitrogen headspace (as indicated from test item data sheet provided by supplier)
Purity: 96.7% prior to study and 96.5% at end of in-life

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: about 8 weeks
- Weight at study initiation: 180-200 g for males and 151-170 g for females (age of 6 weeks)
- Housing:
The animals were initially housed 2 per cage, in polycarbonate cages, with solid bottoms and stainless steel mesh tops and measured ca 48 x 37.5 x 25 cm. A stainless steel food hopper and a polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert measuring ca 48 x 37.5 x 25 cm. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage.
The mated females were transferred to individual solid bottomed cages measuring ca 48 x 37.5 x 25 cm. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters retained this cage type until termination. After mating the males remained singly housed until termination.
- Diet (e.g. ad libitum):ad libitum.
- Water (e.g. ad libitum):ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 39-64%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Amount of vehicle (if gavage): 5 mL per kg body weight
The formulations were prepared at approximately weekly intervals and were used with the 13 days stability period at ambient/dark. The test item was formulated by adding the appropriate volume of vehicle (corn oil) to required amounts of test item and mixed manually and magnetically stirred until a visibly homogeneous solution was obtained.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was undertaken with regard to concentration and homogenity. Samples (3 x 1 mL for Groups 1 and 2, 3 x 0.35 mL for Group 3 and 3 x 0.25 mL for Group 4) were taken from formulations (including Control) prepared for use on Week 1 and Week 6 of the study.
The analysis was undertaken by Toxicology Support Laboratory at Charles River, Edinburgh, using a validated method (Charles River Study No. 426246, Method 2624).

Duration of treatment / exposure:

males: 4 weeks
females: 2 weeks prior to mating then continued until at least Day 4 of lactation.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bodyweight
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bodyweight
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bodyweight
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations:
Prostration
Lethargy
Writhing
Circling
Breathing abnormalities
Gait abnormalities
Tremor
Fasciculation
Convulsions
Biting (of cage components or self mutilating)
Vocalisations
Piloerection

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: twice (ophthalmic examination was undertaken on all animals during Pretrial, all males during Week 4 and all females during lactation)

HAEMATOLOGY: Yes
Samples were obtained during Week 4 for males, and on or after Day 4 of lactation for females.
Blood samples were obtained from 5 males and 5 females from each dose group.
The animals were not deprived of food overnight prior to sampling.
- Parameters checked:
Haemoglobin
Red Blood Cell Count
Haematocrit
White Blood Cell Count
Mean Cell Volume
Mean Cell Haemoglobin
Mean Cell Haemoglobin Concentration
Platelets
Red Cell Distribution Width
Reticulocytes
Differential White Blood Cell Count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Large Unclassified Cells

CLINICAL CHEMISTRY: Yes
Samples were obtained during Week 4 for males, and on or after Day 4 of lactation for females.
Blood samples were obtained from 5 males and 5 females from each dose group.
The animals were not deprived of food overnight prior to sampling.
- Parameters checked:
Urea
Glucose
Aspartate Aminotransferase
Alanine Aminotransferase
Alkaline Phosphatase
Gamma Glutamyl Transferase
Glutamate Dehydrogenase
Lactate Dehydrogenase
Sodium
Potassium
Chloride
Total Protein
Albumin
Globulin – derived
AG Ratio – derived
Cholesterol
Triglycerides
Creatine Phosphokinase
Creatinine
Total Bilirubin
Calcium
Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
5 males and 5 females per group during Week 4 for males (prior to blood sampling) and during lactation for females.
- Battery of functions tested: sensory activity / grip strength / motor activity / Pain Perception / Landing Foot Splay

OTHER:
Coagulation:
Prothrombin Time
Activated Partial Thromboplastin Time
Fibrinogen

Sacrifice and pathology:

The males were sacrificed following 4 weeks of treatment. The females were sacrificed with their litters between Days 5 and Day 6 of lactation.
Adult animals were sacrificed by exposure to carbon dioxide followed by exsanguination.

GROSS PATHOLOGY: Yes
All adults were subject to a detailed necropsy, the necropsy consisted of an external and internal examination which included body orifices (ears, nostrils, mouth, anus, vulva) and cranial, thoracic and abdominal organs and tissues. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number.
HISTOPATHOLOGY: Yes
Histological examination was conducted on 5 males and 5 females from Control and High dose groups, the same animals that were used for Laboratory investigations.

Other examinations:

Bone Marrow Smears

Statistics:

Selected neurotoxicity, body weight, food consumption, haematology and clinical chemistry data were subjected to analysis of variance or the Kruskal-Wallis non-parametric analysis. Organ weights were analysed as above and by one-way analysis of covariance (ANCOVA) using the terminal kill body weight as covariate.
Pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test, or by chi-squared protected z-test (the non-parametric equivalent of Student’s t-test), and were only performed against the Control group.
All statistical tests were two-sided and performed at the 5% significance level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

dermal irritation

food consumption and compound intake

food efficiency

mortality

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD test guideline 422, the repeated dose (sub-acute) NOEL of the registered substance was determine for both sexes to be 1000 mg/kg bw.

Executive summary:

In a GLP-study according to OECD test guideline 422, the repeated dose (28 days) toxicity of the registered substance was determined. Four groups of 10 male and 10 female Sprague-Dawley rats were dosed orally by gavage once daily at levels of 0, 100, 300 and 1000 mg/kg/day. The vehicle was corn oil. The males were treated for 2 weeks prior to mating, through until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation until at least Day 4 of lactation. The animals were monitored daily for any signs of ill health or reaction to treatment, body weight and food consumption performance was monitored and the animals were given opthalmoscopy examinations. Detailed functional observations were performed weekly, with additional functional observations performed on 5 males and 5 females per group on one occasion; Week 4 for males and during lactation for females. Blood samples were also taken from 5 males and 5 females per group for laboratory investigations. Males were sampled during Week 4 and females were sampled during lactation. All adult animals were killed and subjected to a detailed necropsy examination after completion of treatment. Representative samples were taken from all adult animals and fixed in 10% neutral buffered formalin unless stated otherwise, with a selection of tissues being weighed. Histopathology was conducted on tissues from 5 males and 5 females from Control and High dose groups; the same animals that were used for laboratory investigations. At dose levels up to 1000 mg/kg/day, there were no clear toxicological effects of treatment on clinical observations, body weight gain or food consumption performance. The type and distribution of neurotoxicity observations, haematology, coagulation and clinical chemistry parameters did not indicate any association of treatment. There were no necropsy or histology findings that were considered to be related to treatment with the registered substance. Under the conditions of this study, the NOEL (No Observed Effect Level) for both parental and reproductive effects was 1000 mg/kg/day.